Structure and biosynthesis of chrysophysarin A, a plasmodial pigment from the slime mould Physarum polycephalum (Myxomycetes)

Article abstract of DOI:10.1016/S0040-4020(99)01020-0

A yellow optically-active pigment, chrysophysarin A (1), has been isolated from microplasmodia of the slime mould Physarum polycephalum. The structure was established by means of 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configuration

Full text of DOI:10.1016/S0040-4020(99)01020-0

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 363–365
Structure and Biosynthesis of Chrysophysarin A,
a Plasmodial Pigment from the Slime Mould
Physarum polycephalum (Myxomycetes)
*
Sophie Eisenbarth and Bert Steffan
¨
¨
¨
¨
Institut fur Organische Chemie, Ludwig-Maximilians-Universitat Munchen, Butenandtstr. 5-13 (Haus F), D-81377 Munchen, Germany
Received 19 October 1999; accepted 18 November 1999
Abstract—A yellow optically-active pigment, chrysophysarin A (1), has been isolated from microplasmodia of the slime mould Physarum
polycephalum. The structure was established by means of 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configuration
was assigned by the synthesis of N-(3,3-dimethylacryloyl) derivatives of (S)- and (R)-leucine and by comparison of the corresponding CD
spectra. The biosynthesis of the compound was elucidated by feeding labeled acetate to the plasmodia. ᭧ 2000 Elsevier Science Ltd. All
rights reserved.
Introduction
formula C21H30N3O2. In the UV spectrum, the pigment
exhibits absorption maxima at lˆ262 and 355 nm.
For a long time, the myxomycete Physarum polycephalum
has been the subject of intensive research in cell biology,
physiology, genetics and also in chemistry by virtue of its
distinctive feature, which is moving and sporulating under
the influence of light.1 The polyenes physarochrom A,2
physarorubinic acid A and B,3 and the polycephalins B
and C4 have already been isolated from plasmodia of the
intensely yellow-colored slime mould.
Table 1. NMR data of chrysophysarin A (1) in CD3OD/CDCl3 1:1 (v/v)
1H
dH (ppm)
JH,H (Hz)
13C
dC (ppm)
N-1–CH3 (3H)
2-H (1H)
N-3–CH3 (3H)
4-H (1H)
3.88 (s)
8.98 (s)
3.93 (s)
7.69 (s)
N-1–CH3
C-2
N-3–CH3
C-4
34.05 Q
145.49 D
36.44 Q
119.82 D
134.81
C-5
6-H (1H)
7-H (1H)
8-H (1H)
9-H (1H)
10-H (1H)
11-H (1H)
12-H (1H)
13-H (1H)
6.47 (d)
6.98 (dd)
6.60 (m)
6.60 (m)
6.68 (dd)
6.52 (dd)
7.25 (dd)
6.16 (d)
15.7
9.9, 15.7
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
114.72 D
136.42 D
134.82 D
137.20 D
139.30 D
133.62 D
141.30 D
124.89 D
167.85
Chrysophysarin A (1)
10.3, 14.3
11.2, 14.3
11.2, 15.0
15.0
During further chromatographic separation, a substance,
chrysophysarin A (1), with interesting signals in the NMR
spectrum, attracted our attention. Now we wish to report on
the structure and biosynthesis of this compound.
15-H (1H)
16-H (2H)
4.55 (dd)
1.53 (m)
1.60 (m)
1.71 (m)
0.97 (d)
0.93 (d)
4.3, 10.3
58.45 D
C-16
C-17
C-18
C-19
C-20
C-21
39.73 T
25.59 D
21.66 Q
23.40 Q
209.52
Suspension-cultured microplasmodia of P. polycephalum
were completely extracted in the dark. Consecutive stepwise
partition of the red-brown gum and repeated chroma-
tography of the chloroform phase at 4ЊC on Sephadex LH-
20 yielded chrysophysarin A (1) as an amorphous golden-
yellow powder.
17-H (1H)
18-H (3H)
19-H (3H)
6.6
6.6
21-H (3H)
2.21 (s)
26.86 Q
1
The H NMR spectrum displays the signals of a tetraene
system (d 7.25–6.16). Two H resonances at d 8.89 and
The structure of 1 was determined by spectroscopic
analyses. The high resolution mass spectrum (HR-ESI)
shows a molecular ion at 356.23, establishing the molecular
1
7.69 are assigned to an imidazole ring according to their
shift values and coupling constants (145.49 ppm,
Jˆ215 Hz; 119.82 ppm, Jˆ195 Hz). Both nitrogens bear
1
Keywords: slime mould; myxomycetes; Physarum polycephalum; natural
products; polyene; imidazole.
* Corresponding author. Fax: ϩ49-89-2180-7640;
e-mail: bst@cup.unimuenchen.de
methyl groups with H shift values at d 3.88 and 3.93.
1H–1H COSY, HMBC and HMQC experiments connect
signals at d 4.55 and between 1.71 and 0.93 to a leucine
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(99)01020-0

364
S. Eisenbarth, B. Steffan / Tetrahedron 56 (2000) 363–365
Scheme 1. Synthesis of N-(3,3-dimethylacryloyl)-(S)-leucine methylester (2).
residue, whose carboxy group is modified to an acetyl
moiety. Long-range couplings in the 1H and 13C NMR spec-
tra link the imidazole ring to a polyene chain and C-14 over
the amide nitrogen to C-15 of leucine.
Therefore chrysophysarin A has the structure 1.
An incorporation rate of 4.6% into carbon C-21, not only by
feeding [13C2–2-d3]sodium acetate but also by feeding
[2-13C]sodium acetate, shows that the methyl group C-21
originates from a fragmented acetate unit, whose carboxy
group was presumably lost by decarboxylation. A similar
procedure, but without fragmentation, is used to elongate
the carbon chain by the insertion of histidine for the
construction of the polyene system: histidine is transformed
into the corresponding a,b-unsaturated acid and connected
by means of its carboxy group to three acetate units.
To identify the absolute configuration at position C-15 the
N-(3,3-dimethylacryloyl) derivatives of (S)- and (R)-leucine
methylester were synthesized as shown in Scheme 1 and
their CD spectra compared.
Therefore l-leucine, acetate and histidine are proposed as
biosynthetic building blocks for the biosynthesis of chryso-
physarine A (1).
The synthesis started with (S)- and (R)-leucine, respectively,
which were transformed with thionyl chloride/MeOH into
the corresponding methyl esters.5 The (S)- and (R)-leucine
esters were converted with 3,3-dimethylacryloyl chloride
and pyridine to the corresponding amides, which were
obtained as colorless crystals.
Experimental
General
The CD spectra of chrysophysarin and of the (S)-leucine
derivate (2) show an almost parallel course. Therefore
evidence is given for the (S)-configuration in C-15 of
chrysophysarin A (1).
NMR: Bruker AMX2-600 (600.28 MHz for 1H and
150.9 MHz for 13C; chemical shifts are given relative to
the residual solvent signal CD3OD: dHˆ3.35, dCˆ49.0;
CDCl3: dHˆ7.24, dCˆ77.0). – HR-EI MS, HR-ESI MS:
Finnigan MAT 95Q (EI: DI, 210ЊC, 70 eV; ESI: 250ЊC,
2.5 kV). – IR: Perkin-Elmer Spectrum 1000. – UV/VIS:
Biosynthesis
HP 8452A Diode Array Spectrophotometer.
– CD:
Instruments S. A. Jobin Yvon CD-6-Dichrograph. – Analy-
tical TLC: aluminium sheets silica gel 60 F254 (Merck),
0.2 mm, benzene/ethyl formate/formic acid, 10:5:3). –
CC:Sephadex LH-20 (Pharmacia); eluent: MeOH.
For the investigation of the biosynthesis, chrysophysarin A
(1) was isolated, on the one hand from plasmodia, which had
been fed with [2-13C]sodium acetate and, on the other hand,
from plasmodia, which had been grown with [13C2–2-
d3]sodium acetate. The incorporation of the labeled pre-
cursors has been proven by 1D 13C NMR and 13C–13C
COSY NMR spectra. The 13C–13C COSY NMR spectrum
shows the intact acetate units C9–C10, C11–C12 and
C13–C14 with an incorporation rate of 4.5%. The carbons
C-15 and C-20 originate from an additional acetate unit,
whose incorporation rate is 1.4%. The incorporation of
acetate in this position is in accordance with the common
biosynthesis of leucine from valine and acetyl-CoA.6
Culture conditions
Microplasmodia of the yellow wild type strain of
P. polycephalum were cultured under axenic conditions
according to the literature.7 Cells were harvested after 7
days by centrifugation (2500 g) and subsequently washed
with 1 mM Ca(H2PO4)2/10 mM glucose.8

S. Eisenbarth, B. Steffan / Tetrahedron 56 (2000) 363–365
365
Application of labeled precursors
(pHˆ3), water and brine and dried over magnesium sulfate.
The N-(3,3-dimethylacryloyl)-(S)-leucine methylester (2)
was obtained as colorless crystals (yield: 443 mg,
1.95 mmol, 70.9%). – TLC: Rfˆ0.45. – UV/VIS (MeOH):
[2-13C]sodium acetate or [13C2–2-d3]sodium acetate were
added to the standard medium before autoclavation. The
concentration was 50 mg acetate in 100 ml medium
(pHˆ4.75).
lmax (log e)ˆ219 nm (1.09).
– CD (MeOH): lmax
(De)ˆ208 (Ϫ4.41), 221 (Ϫ2.02), 232 (Ϫ2.66), 267
1
(ϩ0.06). – H-NMR (CD3OD/CDCl3 (1:1 (v/v)): dˆ5.75
Extraction and isolation of chrysophysarin A (1)
(m, 1H), 4.52 (m, 1H), 3.73 (s, 3H), 2.12 (d, 3H,
Jˆ1.1 Hz), 1.87 (d, 3H, Jˆ1.1 Hz), 1.69 (m, 1H), 1.62
(m, 2H), 0.96 (d, 3H, Jˆ6.6 Hz), 0.94 (d, 3H, Jˆ6.6 Hz).
All the steps of extraction and isolation were carried out in
the dark because the Physarum pigments are very light
sensitive. The wet biomass (250 g) was completely
extracted with acetone/methanol/chloroform/ethyl acetate
3:2:1:1 (v/v/v/v) in a blender at room temperature, and the
solvent was removed by evaporation. The crude extract
(5.5 g, oil) was partitioned first between 200 ml methanol/
water 9:1 (v/v) and n-hexane ꢀ3×100 ml†: The combined
n-hexane phases were washed once with methanol/water
9:1 (100 ml). The methanol/water layers were combined,
the solvent was evaporated, and the residue was dissolved
again in 200 ml of water and extracted with chloroform
ꢀ3×100 ml†: Repeated chromatography of the chloroform
phase (337 mg) at 4ЊC on Sephadex LH-20 with methanol
ꢀ80 cm×6 cm; i.d.) yielded a faint yellow fraction, which
gave on evaporation pure chrysophysarin A (1) (26 mg,
0.000104%) as an amorphous golden-yellow powder.
–
13C NMR (CD3OD/CDCl3, 1:1 (v/v)): dˆ174.71, 168.77,
152.27, 118.49, 52.45, 51.38, 41.18, 27.31, 25.42, 23.07,
21.85, 20.03. – IR (KBr): n~ˆ3435 (m), 3302 (s), 3072
(w), 2958 (m), 2871 (m), 1745 (s), 1664 (s), 1634 (s),
1545 (s), 1270 (m), 1215 (s), 1183 (s), 1153 (m), 980
(w). – HR-EI MS: m/zˆ227.1518 (227.1521 calcd. for
C12H21NO3) [Mϩ].
Acknowledgements
The authors gratefully acknowledge the support of the
Deutsche Forschungsgemeinschaft (SFB 369). We would
¨
also like to thank Dr. Wolfgang Marwan, Universitat
Freiburg, for a suspension culture of Physarum poly-
cephalum.
Chrysophysarin A (1). TLC: Rfˆ0.12. – UV/VIS (MeOH):
lmax (log e)ˆ262 nm (0.40), 341 (1.28), 355 (1.55), 372
(1.15). – CD (MeOH): lmax (De)ˆ206 (Ϫ0.63), 223
(ϩ0.08), 250 (Ϫ0.17), 297 (ϩ0.03), 354 (ϩ0.23). – 1H
NMR and 13C NMR see Table 1. – IR (KBr): n~ ˆ 3400
(s), 2923 (s), 2852 (m), 1711 (w), 1619 (s), 1466 (m),
1396 (m), 1016 (w), 951 (w). – HR-ESI MS: m/zˆ
356.2327 (356.2338 calcd. for C21H30N3O2) [Mϩ].
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